Methods for production of capsular polysaccharide protein conjugates from streptococcus pneumoniae serotype 19f

ABSTRACT

The present invention provides a method of producing a polysaccharide-protein conjugate with capsular polysaccharide from Streptococcus pneumoniae serotype 19F conjugated to a carrier protein. The method includes a prolonged incubation step prior to filtration to remove free polysaccharide.

FIELD OF INVENTION

The present invention relates to a method of producing a pneumococcalserotype 19F capsular polysaccharide protein conjugate. In particular,the method provides for greater stability of polysaccharide proteinconjugates having pneumococcal serotype 19F by providing a prolongedincubation time after conjugation and prior to filtration. The inventionalso relates to the preparation of a multivalent pneumococcal conjugatevaccine comprising stable serotype 19F polysaccharide protein conjugate.

BACKGROUND OF THE INVENTION

Polysaccharide-protein conjugate vaccines, comprising bacterial capsularpolysaccharides conjugated to carrier proteins have been developed andadditional ones are in development. Examples of developed conjugatevaccines include the Haemophilus influenzae type b (Hib) conjugatevaccine (e.g., HIBTITER®) as well as conjugate vaccines againstStreptococcus pneumoniae (e.g., PREVNAR® and PREVNAR 13®) and Neisseriameningitidis (e.g., MENJUGATE®).

Upon the conjugation of a polysaccharide antigen to a carrier protein,the reaction mixture can be purified to remove free polysaccharide thathas no protein conjugated thereto, free carrier protein that has nopolysaccharide antigen conjugated thereto, and low molecular weightpolysaccharide protein conjugates. Various methods for the purificationof free polysaccharide, free protein, and low molecular weightconjugates are known in the art, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. See, e.g., InternationalPatent Application Publication No. WO00/38711, U.S. Pat. No. 6,146,902,and Lei et al., 2000, Dev. Biol. 103:259-264.

There is a continuing need for improved methods of producing stablepolysaccharide protein conjugates and purifying polysaccharide proteinconjugates from impurities such as free polysaccharide and low molecularweight conjugates.

SUMMARY OF THE INVENTION

The present invention provides methods for the production andpurification of a polysaccharide-protein conjugate comprisingStreptococcus pneumoniae serotype 19F capsular polysaccharide covalentlylinked to a carrier protein from a mixture comprisingpolysaccharide-protein conjugate and free polysaccharide, the methodcomprising the steps of:

a) incubating said mixture for a minimum of 6 hours, at a temperatureranging from 2−30° C., in a buffer having a pH in the range of 5.0 to9.0; and

b) performing size separation under conditions that allow removal offree polysaccharide.

In certain embodiments, the size separation uses a nominal molecularweight cut off (NMWCO) membrane of from 100 to 500 kDa whereby thepolysaccharide-protein conjugate is retained in the retentate.

In certain embodiments, the methods further comprise

c) collecting the polysaccharide-protein conjugate.

The methods described herein are applicable to carrier proteinsincluding but not limited to tetanus toxoid, diphtheria toxoid, andCRM₁₉₇. In certain embodiments of the invention, the carrier protein isCRM₁₉₇.

In certain embodiments, the retained polysaccharide-protein conjugatehas an average molecular weight of 600 kDa or more, or 1000 kDa or more.

In certain embodiments, the pH of the incubation is in the range from5.8 to 7.0. The buffer employed in the methods of the invention can beselected from a phosphate buffer, histidine, or TRIS and have a pH inthe range of pH 5.8 to 7.0. In one embodiment, the buffer has a pH of7.0.

In certain embodiments, the temperature of incubation is controlled inthe range of 4 -25° C.

In certain embodiments, the size separation is by size-exclusionchromatography, bind/elute chromatography, or wide-pore ultrafiltration.In one aspect, the size separation is by wide-pore ultrafiltration witha membrane having a NMWCO of 100 kDa to 300 kDa.

In certain embodiments, the incubation proceeds for at least 12 hours orat least 20 hours, for example, between 108 to 132 hours.

The present invention also provides methods for formulating thepolysaccharide-protein conjugate with one or more additionalpolysaccharide-protein conjugates from a different serotype. In oneembodiment, the methods further comprise formulating with an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Serotype 19F conjugate stability as measured by freepolysaccharide (Ps) change at 4° C. Free Ps content for two serotype19F-CRM₁₉₇ conjugate lots as a function of time at pH 7.0 and 4° C.Serotype 19F conjugate lot A was not incubated, lot B was incubated at22° C. for approximately 5 days prior to wide-pore ultrafiltrationpurification. See Table 1.

FIG. 2: Serotype 19F conjugate accelerated stability as measured by freePs change at 25° C. Free Ps content for three serotype 19F-CRM₁₉₇conjugate lots as a function of time at pH 5.8 and 25° C. Serotype 19Fconjugate lot A was not incubated, lot B was incubated at 22° C. forapproximately 5 days in 10 mM histidine, 150 mM sodium chloride, pH 7.0prior to wide-pore ultrafiltration purification. See Table 1. Serotype19F lot C was incubated at 22° C. for approximately 5 days at pH 7.0 in25 mM potassium phosphate, 150 sodium chloride prior to wide-poreultrafiltration purification.

FIG. 3: Free Ps content in two serotype 19F conjugate lots B and C(described in Table 1 and FIG. 2) as a function of time during thein-process incubation step to improve drug substance (DS) and drugproduct (DP) stability. Free Ps generated during incubation is clearedin subsequent wide-pore ultrafiltration step.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that traditionalmethods, including ultrafiltration, were insufficient to provide astable polysaccharide-protein conjugate for S. pneumoniae serotype 19F.Specifically, the 19F conjugate was found to be unstable and had reducedpotency. As shown in the Examples, Applicants' work demonstrated thatafter conjugation of serotype 19F polysaccharide to CRM₁₉₇, theconjugated 19F polysaccharide is prone to degradation, resulting inproduct that has elevated free polysaccharide levels and reducedconjugate size. A plateau of between 25-30% free polysaccharide wasobtained whether at 4° C. (after approximately 3 months) or 22° C-25° C.(after approximately 5-7 days).

Without being bound by any theory, this degradation event is believed tobe due to the presence of labile sites on the 19F polysaccharide. Byallowing degradation of the labile sites to occur, through a prolongedincubation time after conjugation and prior to purification, theresulting conjugate can be stabilized so that the free polysaccharidecontent, conjugate size, and conjugate potency do not change over time.

Accordingly, the present invention describes a method for the productionof a polysaccharide-protein conjugate comprising Streptococcuspneumoniae serotype 19F covalently linked to a carrier protein from amixture comprising polysaccharide-protein conjugate and freepolysaccharide by incubating said mixture for 6 hours or longer, 12hours or longer, 20 hours or longer or 24 hours or longer in anappropriate buffer (such as phosphate, histidine, or any buffer with apKa in the range of 6-9); and performing a purification step, such aswide-pore ultrafiltration, under conditions that allow removal of freepolysaccharide, and optionally collecting the polysaccharide-proteinantigen from the retentate. The free polysaccharide may optionally becollected from the ultrafiltration permeate and re-used in a conjugationreaction, if desired.

The conjugation reaction mixture may comprise polysaccharideantigen-carrier protein conjugates and free polysaccharide. The mixturemay also contain free carrier protein, low molecular weight conjugatesand other proteins. The method of the invention providespolysaccharide-protein conjugates of higher stability.

The invention further provides a method of preparing an immunogeniccomposition by mixing the purified polysaccharide-protein conjugate fromserotype 19F with additional polysaccharide-protein conjugates fromadditional S. pneumoniae serotypes.

As used herein, the term “comprises” when used with the immunogeniccomposition of the invention refers to the inclusion of any othercomponents (subject to limitations of “consisting of” language for theantigen mixture), such as adjuvants and excipients. The term “consistingof” when used with the multivalent polysaccharide-protein conjugatemixture refers to a mixture having those particular S. pneumoniaepolysaccharide protein conjugates and no other S. pneumoniaepolysaccharide protein conjugates from a different serotype.

As used herein, the phrase “drug product” refers to the formulated blendof polysaccharide-carrier protein conjugates from two or more serotypes.

As used herein, the phrase “drug substance” refers to the individualpolysaccharide-carrier protein conjugate from a given serotype.

As used herein, the phrase “polysaccharide-protein conjugate fromserotype” refers to a conjugate having a S. pneumoniae capsularpolysaccharide obtained from the specified serotype, e.g., 19F, and acarrier protein, e.g., CRM₁₉₇.

As used herein, ranges used for, for example, pH and temperature, aremeant to be inclusive. For example, a pH range from 5.0 to 9.0 is meantto include a pH of 5.0 and a pH of 9.0. Similarly, a temperature rangefrom 4 to 25° C. is meant to include the outer limits of the range,i.e., 4° C. and 25° C.

Streptococcus pneumoniae Capsular Polysaccharides

Capsular polysaccharides from Streptococcus pneumoniae, includingserotype 19F, can be prepared by standard techniques known to thoseskilled in the art. For example, polysaccharides can be isolated frombacteria and may be sized to some degree by known methods (see, e.g.,European Patent Nos. EP497524 and EP497525); and preferably bymicrofluidisation accomplished using a homogenizer or by chemicalhydrolysis. In certain techniques, S. pneumoniae strains correspondingto each polysaccharide serotype are grown in a soy-based medium. Theindividual polysaccharides are then purified through standard stepsincluding centrifugation, precipitation, and ultrafiltration. See, e.g.,U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat. No.5,847,112. Polysaccharides can be sized in order to reduce viscosityand/or to improve filterability and the lot-to-lot consistency ofsubsequent conjugated products. Capsular polysaccharides can also beprepared from one or more of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 20,22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F, or 38 for inclusion inmultivalent pneumococcal polysaccharide protein conjugate vaccines.

Carrier Protein

In a particular embodiment of the present invention, CRM₁₉₇ is used asthe carrier protein. CRM₁₉₇ is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin. In one embodiment, it is isolated from cultures ofCorynebacterium diphtheria strain C7 (β197) grown in casamino acids andyeast extract-based medium. In another embodiment, CRM₁₉₇ is preparedrecombinantly in accordance with the methods described in U.S. Pat. No.5,614,382. Typically, CRM₁₉₇ is purified through a combination ofultra-filtration, ammonium sulfate precipitation, and ion-exchangechromatography. In some embodiments, CRM₁₉₇ is prepared in Pseudomonasfluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego,Calif.).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment Cof TT, pertussis toxoid, cholera toxoid (e.g., as described inInternational Patent Application Publication No. WO 2004/083251), E.coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.Bacterial outer membrane proteins such as outer membrane complex c(OMPC), porins, transferrin binding proteins, pneumococcal surfaceprotein A (PspA; See International Application Patent Publication No. WO02/091998), pneumococcal surface adhesin protein (PsaA), C5a peptidasefrom Group A or Group B streptococcus, or Haemophilus influenzae proteinD, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (SeeInternational Patent Application Publication No. WO 04/081515) ordPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions of Phtproteins for example PhtDE fusions, PhtBE fusions (See InternationalPatent Application Publication Nos. WO 01/98334 and WO 03/54007), canalso be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0594 610 B), or immunologically functional equivalents thereof, syntheticpeptides (See European Patent Nos. EP0378881 and EP0427347), heat shockproteins (See International Patent Application Publication Nos. WO93/17712 and WO 94/03208), pertussis proteins (See International PatentApplication Publication No. WO 98/58668 and European Patent No.EP0471177), cytokines, lymphokines, growth factors or hormones (SeeInternational Patent Application Publication No. WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (See Falugi et al., 2001, Eur JImmunol 31:3816-3824) such as N19 protein (See Baraldoi et al., 2004,Infect Immun 72:4884-7), iron uptake proteins (See International PatentApplication Publication No. WO 01/72337), toxin A or B of C. difficile(See International Patent Publication No. WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can be used, such as CRM₁₇₆, CRM₂₂₈, CRM₄₅ (Uchida etal., 1973, J Biol Chem 218:3838-3844); CRM₉, CRM₄₅, CRM₁₀₂, CRM₁₀₃ andCRM₁₀₇ and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragments disclosed in U.S.Pat. No. 5,843,711.

Polysaccharide-Protein Conjugation

The purified polysaccharides are typically chemically activated tointroduce functionalities capable of reacting with the carrier protein.Once activated, each capsular polysaccharide is separately conjugated toa carrier protein to form a glycoconjugate. The polysaccharideconjugates may be prepared by known coupling techniques.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein are achieved by meansdescribed in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506. Briefly,the pneumococcal polysaccharide is reacted with a periodate-basedoxidizing agent such as sodium periodate, potassium periodate, orperiodic acid resulting in random oxidative cleavage of vicinal hydroxylgroups to generate reactive aldehyde groups.

Direct aminative coupling of the oxidized polysaccharide to primaryamine groups on the protein carrier (mainly lysine residues) can beaccomplished by reductive amination. For example, conjugation is carriedout by reacting a mixture of the activated polysaccharide and carrierprotein with a reducing agent such as sodium cyanoborohydride in thepresence of nickel. The conjugation reaction may be carried out inaqueous solution or in an organic solvent such as dimethylsulfoxide(DMSO). See, e.g., US2015/0231270 Al, EP 0471 177 B1, US2011/0195086 A1.At the conclusion of the conjugation reaction, unreacted aldehydes areoptionally reduced by addition of a strong reducing agent, such assodium borohydride.

In one embodiment, prior to formulation, each pneumococcal capsularpolysaccharide antigen is individually purified from S. pneumoniae,activated to form reactive aldehydes, and then covalently conjugated toa carrier protein using reductive amination with sodium cyanoboroydridein the presence of nickel. Nickel forms complexes with residual,interfering cyanide from the sodium cyanoborohydride reducing agent usedfor reductive amination. Thus, nickel is used in the methods of theinvention for greater conjugation reaction efficiency and to aid in freecyanide removal.

Transition metals are known to form stable complexes with cyanide andare known to improve reductive methylation of protein amino groups andformaldehyde with sodium cyanoborohydride. See Gidley et al., Biochem J.1982, 203: 331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190.However, Applicants surprisingly found that by complexing residual,interfering cyanide, the addition of nickel increases the consumption ofprotein during the conjugation of and leads to formation of larger,potentially more immunogenic conjugates.

Variability in free cyanide levels in commercial sodium cyanoborohydridereagent lots may lead to inconsistent conjugation performance, resultingin variable conjugate attributes, including molecular mass andpolysaccharide-to-protein ratio. The addition of nickel to theconjugation reaction reduces the level of free cyanide and thus improvesthe degree of lot-to-lot conjugate consistency.

In another embodiment, the conjugation method may employ activation ofpolysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may be coupleddirectly to an amino group on the carrier protein.

In an alternative embodiment, a reactive homobifunctional orheterobifunctional group may be introduced on the activatedpolysaccharide by reacting the cyanate ester with any of severalavailable modalities. For example, cystamine or cysteamine may be usedto prepare a thiolated polysaccharide which could be coupled to thecarrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). Such conjugates are described in International PatentApplication Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094;and Chu et al., 1983, Infect. Immunity 40:245-256.

Other suitable conjugation methods use carbodiimides, hydrazides, activeesters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS,EDC, TSTU. Many are described in International Patent ApplicationPublication No. WO 98/42721. Conjugation may involve a carbonyl linkerwhich may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (See Bethell et al., 1979, J. Biol. Chem.254:2572-4; Hearn et al., 1981, J. Chromatogr. 218:509-18) followed byreaction with carrier protein to form a carbamate linkage. Thischemistry consists of reduction of the anomeric terminus of acarbohydrate to form a primary hydroxyl group followed by reaction ofthe primary hydroxyl with CDI to form a carbamate intermediate andsubsequent coupling to protein carrier amino groups. The reaction mayrequire optional protection/deprotection of other primary hydroxylgroups on the saccharide.

Following conjugation, the polysaccharide-protein conjugates arepurified to remove excess conjugation reagents as well as residual freeprotein and free polysaccharide by one or more of any techniques wellknown to the skilled artisan, including concentration/diafiltrationoperations, ultrafiltration, precipitation/elution, columnchromatography, and depth filtration. See, e.g., U.S. Pat. No.6,146,902.

For serotype 19F, it was discovered that there was a rapid decline inpotency over time indicating relative instability of the conjugatedmaterial. This was found to be associated with an increase in freepolysaccharide and a decrease in conjugate size. In order to overcomethese issues, it was discovered that an incubation period after theconjugation prior to purification using, for example, a wide-poreultrafiltration step to remove free polysaccharide improved thestability of the polysaccharide protein conjugate. This increase in freepolysaccharide is believed to be due to the presence of labile sites onthe 19F polysaccharide. By incubating 19F polysaccharide afterconjugation (and prior to purification), the portion of 19Fpolysaccharide prone to degradation (˜25-30%) can be allowed to degradeand be removed by a filtration step.

The incubation conditions should be chosen to allow the 19Fpolysaccharide to degrade through its labile sites, but not includeconditions which would degrade the polysaccharide through othermechanisms, such as excessively high or low pH. As shown in theExamples, degradation of non-incubated 19F conjugate to freepolysaccharide reached the plateau of about 30% at about 3 months at 4°C. and at about 7 days at 25° C. It is readily apparent that time andtemperature (and pH) can be varied under a number of differentconditions to obtain the maximum degradation of that portion of theconjugate containing labile sites.

The incubation can take for as long as five days or longer, for example,5 to 7 days, to achieve optimal removal of free Ps. However, significantreduction for free Ps can be achieved in as little as one day.Accordingly, the present invention provides methods where the incubationoccurs for a minimum of 1, 3, 6, 12, 18, 24, 36, 48, 60 or 72 hours. Theincubation can occur for up to 60, 72, 84, 96, 108, 120, or 132 hours.The present invention encompasses all combination of these incubationtimes including for example, 6 to 96 hours, 24 to 84 hours, 48 to 60hours, as well as 72 hours to 132 hours, 96 hours to 132 hours and 108hours to 132 hours. It would be expected that times greater than 132hours, e.g., 150 hours, 180 hours, 200 hours, 240 hours and greater,could be used with minimal effect on the degradation. Generally, ahigher degradation rate will be seen at higher temperatures so whenhigher temperatures are used, the incubation time can be shorter.

The incubation preferably takes place in the buffer used for conjugationor following conjugation. The buffer can be selected from histidine,phosphate, TRIS or any buffer with a pH in the range of 6.0-9.0, 6.0 to8.5, or 6.5 to 7.5. In certain embodiments, the buffer has a pH of 6.0,6.5, 7.0, 7.5, 8.0, 8.5 or 9.0.

The buffer optionally further contains a salt selected from sodium andpotassium chloride. Ranges of salt concentrations are from 0-500 mM. Inone embodiment, the buffer is 10 mM histidine or 25 mM potassiumphosphate and further includes 150 mM sodium chloride.

The pH of the incubation can occur between a pH from 5.0 to 9.0, 5.8 to7.0, or 7.0±0.2.

The temperature of the incubation can occur between 2-30° C., 4-25° C.,15-25° C., or 20-25° C. As discussed above, at higher temperatures, thekinetics of degradation occurs more quickly. With higher temperatures,the incubation times can be shorter. Conversely, at lower temperatures,the kinetics of degradation occurs more slowly. With lower temperatures,the incubation times are generally longer.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates are purified (enriched withrespect to the amount of polysaccharide-protein conjugate of the desiredsize range, e.g., by removing free polysaccharide) by size separationusing one or more of a variety of techniques. Examples of thesetechniques are well known to the skilled artisan and includeconcentration/diafiltration operations, ultrafiltration includingwide-pore ultrafiltration, precipitation/elution, column chromatographyincluding size-exclusion and bind/elute chromatography, and depthfiltration. See, e.g., U.S. Pat. No. 6,146,902. The appropriatemolecular weight cut off can be selected from 100 kDa to 500 kDa, e.g.,100 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa or 500 kDa. In particularembodiments, the size separation is accomplished by wide-poreultrafiltration with a membrane having a MWCO of 100 kDa to 300 kDa.

Purification not only removes free polysaccharide but can also removelow molecular weight conjugates thereby increasing the overall averagemolecular weight of the polysaccharide-protein conjugates. In someembodiments, the average molecular weight of the retained conjugates is600 kDa, 700 kDa, 800 kDa, 900 kDa, or 1000 kDa or more.

The polysaccharide protein conjugate can then be collected from theretentate on the filter or column using standard techniques.

Following the purification step, the product is typically 0.2-micronfiltered in preparation for formulation.

Pharmaceutical/Vaccine Compositions

The serotype 19F conjugate prepared using the methods of the inventioncan be used in compositions, including pharmaceutical, immunogenic andvaccine compositions, comprising, consisting essentially of, oralternatively, consisting of any polysaccharide serotype combinationstogether with a pharmaceutically acceptable carrier and an adjuvant. Forexample, the compositions can comprise, consist essentially of, orconsist of 2 to 35, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 distinctpolysaccharide-protein conjugates, wherein each of the conjugatescontains a different capsular polysaccharide conjugated individually toone or more carrier proteins, and wherein the capsular polysaccharides(in addition to 19F) further comprise at least one of serotypes 1, 2, 3,4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C,16F, 17F, 18C, 19A, 20, 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F, or 38 ofStreptococcus pneumonia, together with a pharmaceutically acceptablecarrier and an adjuvant. In certain embodiments, the carrier protein isCRM₁₉₇.

After the individual glycoconjugates are prepared, purified andfiltered, as described above, they are compounded using standardtechniques to formulate the immunogenic composition of the presentinvention. These pneumococcal conjugates are prepared by separateprocesses and bulk formulated into a single dosage formulation.

Formulation of the polysaccharide-protein conjugates of the presentinvention can be accomplished using art-recognized methods. Forinstance, 9, 11, 13, 15 or more individual pneumococcal conjugates canbe formulated with a physiologically acceptable vehicle to prepare thecomposition. Examples of such vehicles include, but are not limited to,water, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol) and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated inL-histidine buffer with sodium chloride.

As defined herein, an “adjuvant” is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan. Suitable adjuvants to enhance effectiveness of thecomposition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting of3-O-deaylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylaminol]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646); and

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143:489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,alhydrogel, Superfos, Amphogel, aluminum (III) hydroxide, aluminumhydroxyphosphate sulfate (Aluminum Phosphate Adjuvant (APA)), amorphousalumina, trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel or Superfos of Denmark/Accurate Chemical and Scientific Co.,Westbury, N.Y.) is used to adsorb proteins. Adsorption of protein isdependent, in another embodiment, on the pI (Isoelectric pH) of theprotein and the pH of the medium. A protein with a lower pI adsorbs tothe positively charged aluminum ion more strongly than a protein with ahigher pI. Aluminum salts may establish a depot of antigen that isreleased slowly over a period of 2-3 weeks, be involved in nonspecificactivation of macrophages and complement activation, and/or stimulateinnate immune mechanism (possibly through stimulation of uric acid).See, e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.

In certain embodiments, monovalent bulk aqueous conjugates are typicallyblended together and diluted to target 8 μg/mL for all serotypes except6B, if used, which will be diluted to target 16 μg/mL. Once diluted, thebatch will be filter sterilized, and an equal volume of aluminumphosphate adjuvant added aseptically to target a final aluminumconcentration of 250 μg/mL. The adjuvanted, formulated batch willtypically be filled into single-use, 0.5 mL/dose vials.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21:4297. In another embodiment,any other art-accepted definition of the terms is intended.CpG-containing oligonucleotides include modified oligonucleotides usingany synthetic internucleoside linkages, modified base and/or modifiedsugar.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93;Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006,Crit Rev Ther Drug Carrier Syst. 23:89-110.

Administration/Dosage

The compositions and formulations of the present invention can be usedto protect or treat a human susceptible to infection, e.g., apneumococcal infection, by means of administering the vaccine via asystemic or mucosal route. In one embodiment, the present inventionprovides a method of inducing an immune response to a S. pneumoniaecapsular polysaccharide conjugate, comprising administering to a humanan immunologically effective amount of an immunogenic composition of thepresent invention. In another embodiment, the present invention providesa method of vaccinating a human against a pneumococcal infection,comprising the step of administering to the human an immunologicallyeffective amount of an immunogenic composition of the present invention.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivity of a microbe, e.g., S. pneumonia, during asubsequent challenge.

The composition of the invention can be used for the prevention and/orreduction of primary clinical syndromes caused by microbes, e.g., S.pneumonia, including both invasive infections (meningitis, pneumonia,and bacteremia), and noninvasive infections (acute otitis media, andsinusitis).

Administration of the compositions of the invention can include one ormore of: injection via the intramuscular, intraperitoneal, intradermalor subcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. In one embodiment,intranasal administration is used for the treatment of pneumonia orotitis media (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage).

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, for polysaccharide-based conjugates, each dose will comprise0.1 to 100 μg of each polysaccharide, particularly 0.1 to 10 μg, andmore particularly 1 to 5 μg. For example, each dose can comprise 100,150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7,7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60,70, 80, 90, or 100 μg.

In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30,50, 70, 100, 125, 150, 200, 300, 500, or 700 μg, or 1, 1.2, 1.5, 2, 3, 5mg or more. In yet another embodiment, the dose of alum salt describedabove is per μg of recombinant protein.

The compositions described herein are preferably administered to a humansubject. In certain embodiments, the human patient is an infant (lessthan 1 year of age), toddler (approximately 12 to 24 months), or youngchild (approximately 2 to 5 years). In other embodiments, the humanpatient is an elderly patient (>65 years). The compositions of thisinvention are also suitable for use with older children, adolescents andadults (e.g., aged 18 to 45 years or 18 to 65 years).

A composition described herein can be administered as a singleinoculation. The vaccine can be administered twice, three times or fourtimes or more, adequately spaced apart. For example, the composition maybe administered at 1, 2, 3, 4, 5, or 6 month intervals or anycombination thereof. The immunization schedule can follow thatdesignated for pneumococcal vaccines. For example, the routine schedulefor infants and toddlers against invasive disease caused by S.pneumoniae is 2, 4, 6 and 12-15 months of age. Thus, in a preferredembodiment, the composition is administered as a 4-dose series at 2, 4,6, and 12-15 months of age.

The compositions may also include one or more proteins from S.pneumoniae. Examples of S. pneumoniae proteins suitable for inclusioninclude those identified in International Patent Application PublicationNos. WO 02/083855 and WO 02/053761.

Formulations

The compositions described herein can be administered to a subject byone or more method known to a person skilled in the art, such asparenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly.

In one embodiment, compositions can be administered via epidermalinjection, intramuscular injection, intravenous, intra-arterial,subcutaneous injection, or intra-respiratory mucosal injection of aliquid preparation. Liquid formulations for injection include solutionsand the like.

The composition can be formulated as single dose vials, multi-dose vialsor as pre-filled syringes.

Compositions described herein can be administered orally, and would thusformulated in a form suitable for oral administration, i.e., as a solidor a liquid preparation. Solid oral formulations include tablets,capsules, pills, granules, pellets and the like. Liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor non-aqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However it is often preferred that a pharmaceutical composition forinfusion or injection is essentially isotonic, when it is administrated.Hence, for storage the pharmaceutical composition may preferably beisotonic or hypertonic. If the pharmaceutical composition is hypertonicfor storage, it may be diluted to become an isotonic solution prior toadministration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to sodium chloride (NaCl),calcium chloride (CaCl₂), potassium chloride (KCl) and magnesiumchloride (MgCl₂). Examples of non-ionic isotonic agents include but arenot limited to mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may for example be selected from the group consisting ofTRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, histidine, glycine, succinate andtriethanolamine buffer.

The buffer may furthermore for example be selected from USP compatiblebuffers for parenteral use, in particular, when the pharmaceuticalformulation is for parenteral use. For example the buffer may beselected from the group consisting of monobasic acids such as acetic,benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic,adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,polybasic acids such as citric and phosphoric; and bases such asammonia, diethanolamine, glycine, triethanolamine, and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The DP formulations described herein may also contain a surfactant.Preferred surfactants include, but are not limited to: thepolyoxyethylene sorbitan esters surfactants (commonly referred to as theTweens); copolymers of ethylene oxide (EO), propylene oxide (PO), and/orbutylene oxide (BO), sold under the DOWFAX™ tradename, such as linearEO/PO block copolymers; octoxynols, which can vary in the number ofrepeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (TritonX-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate.

Mixtures of surfactants can be used, e.g. PS80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Analytical

For pneumococcal conjugate vaccines, release and stability testing isimportant for quality control. Each polysaccharide-protein conjugate ina commercial lot is tested against a reference standard to ensure theappropriate dose and/or potency. Stable reference standards ensureconsistent and robust testing results for both release and stabilitytesting. If a reference standard degrades over time, the test resultsgenerated in a relative assay, such as those often used to measurepotency, will drift over time.

Applicants' work with 19F showed that additional incubation prior topurification improved its stability. Thus, the use of 19F conjugate madeby the process described herein as a reference standard would betterensure consistent test results over time and enable better methodprecision and accuracy. If 19F conjugate not subject to this additionalincubation step were used as a reference standard, the degradation seenwith 19F could introduce systematic bias into the test results.

Thus, in certain embodiments, the present invention is also directed topolysaccharide protein conjugate immunoassays for measuring dose and/orpotency in a pneumococcal conjugate vaccine manufacturing lot using 19Fconjugate as a reference standard. Such immunoassays include thesandwich ELISA described in the Analytical section of the Examples.

Having described various embodiments of the invention with reference tothe accompanying description and drawings, it is to be understood thatthe invention is not limited to those precise embodiments, and thatvarious changes and modifications may be effected therein by one skilledin the art without departing from the scope or spirit of the inventionas defined in the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLES Analytical Methods Molecular Weight and Concentration Analysisof Conjugates Using HP SEC/UV/MALS/RI Assay

Conjugate samples were injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection was accomplished withultraviolet (UV), multi- angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration was calculatedfrom A₂₈₀ (absorbance at 280 nm) using an extinction coefficient.Polysaccharide concentration was deconvoluted from the RI signal(contributed by both protein and polysaccharide) using the dn/dc factorswhich are the change in a solution's refractive index with a change inthe solute concentration reported in mL/g. Average molecular weight ofthe samples were calculated by Astra software (Wyatt TechnologyCorporation, Santa Barbara, Calif.) using the measured concentration andlight scattering information across the entire sample peak. There aremultiple forms of average values of molecular weight for polydispersedmolecules.

For example, number-average molecular weight Mn, weight-averagemolecular weight Mw, and z-average molecular weight Mz (Molecules, 2015,20, 10313-10341). Unless specified, the molecular weights areweight-average molecular weight.

Free Polysaccharide Testing

Free polysaccharide (polysaccharide that is not conjugated with CRM₁₉₇)in conjugate sample was measured by first precipitating free protein andconjugates with deoxycholate (DOC) and hydrochloric acid. Precipitateswere then filtered out and the filtrates were analyzed for freepolysaccharide concentration by HPSEC/UV/MALS/RI. Free polysaccharide iscalculated as a percentage of total polysaccharide measured byHPSEC/UV/MALS/RI.

Total Polysaccharide ELISA

The Total Polysaccharide (Ps) Sandwich ELISA is a multi-valentimmunoassay intended to measure the total amount of polysaccharide indrug product samples. Ps is captured and detected by serotype-specificantibodies, and the polysaccharide content is compared relative to astandard by parallel line analysis of dilution curves. Serotype-specificantibodies are first coated directly on the microtiter plate. After ablocking step, dilution curves of standards and samples are applied tothe coated microtiter plates. Immobilized polysaccharides are detectedwith a mix of serotype-specific antibodies and a secondary antibodyconjugated to alkaline phosphatase (AP Conjugate). A fluorescent signalis developed with 4-Methylumbelliferyl phosphate (4-MuP).

Example 1 Preparation of S. Pneumoniae Capsular Polysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP0497524. Isolates ofpneumococcal subtypes are available from the American Type CultureCollection (Manassas, Va.). The bacteria are identified as encapsulated,non-motile, Gram-positive, lancet-shaped diplococci that arealpha-hemolytic on blood-agar. Subtypes can be differentiated on thebasis of Quelling reaction using specific antisera. See, e.g., U.S. Pat.No. 5,847,112.

Cell banks representing each of the S. pneumococcus serotypes presentwere obtained from the Merck Culture Collection (Rahway, N.J.) in afrozen vial. A thawed seed culture was transferred to the seed fermentorcontaining a pre-sterilized growth media appropriate for S. pneumoniae.The culture was grown in the seed fermentor with temperature and pHcontrol. The entire volume of the seed fermentor was transferred to aproduction fermentor containing pre-sterilized growth media. Theproduction fermentation was the final cell growth stage of the process.Temperature, pH, and the agitation rate were controlled.

The fermentation process was terminated via the addition of aninactivating agent. After inactivation, the batch was transferred to theinactivation tank where it was held at controlled temperature andagitation. Cell debris was removed using a combination of centrifugationand filtration. The batch was ultrafiltered and diafiltered. The batchwas then subjected to solvent-based fractionations that removeimpurities and recover polysaccharide.

Example 2 Conjugation of Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19A, 19F, 22F, 23F, and 33F to CRM₁₉₇ using Reductive Amination inAqueous Solution

The different serotype polysaccharides were individually conjugated topurified CRM₁₉₇ carrier protein using a common process flow.Polysaccharide was dissolved, size reduced, chemically activated andbuffer-exchanged by ultrafiltration. Purified CRM₁₉₇ was then conjugatedto the activated polysaccharide utilizing NiCl₂ (2 mM) in the reactionmixture, and the resulting conjugate was purified by ultrafiltrationprior to a final 0.2-micron filtration. Several process parameterswithin each step, such as pH, temperature, concentration, and time werecontrolled to serotype-specific values as described in the sectionsbelow.

Polysaccharide Size Reduction and Ativation

Purified pneumococcal capsular Ps powder was dissolved in water, and allserotypes, except serotype 19A, were 0.45-micron filtered. Allserotypes, except serotype 19A, were homogenized to reduce the molecularmass of the Ps. Serotype 19A was not size reduced due to its relativelylow starting size. Homogenization pressure and number of passes throughthe homogenizer were controlled to serotype-specific targets (150-1000bar; 4-7 passes) to achieve a serotype-specific molecular mass.Size-reduced polysaccharide was 0.2-micron filtered and thenconcentrated and diafiltered against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to a serotype-specifictemperature (4-22° C.) and pH (4-5) with sodium acetate buffer tominimize Ps size reduction during the activation step. Polysaccharideactivation was performed via periodate oxidation. For serotype 4, priorto activation, the batch was incubated at approximately 50° C. and pH4.0 to partially deketalize the Ps. Ps activation was initiated with theaddition of a sodium metaperiodate solution. The amount of sodiummetaperiodate added was serotype-specific, ranging from approximately0.1 to 0.5 moles of sodium metaperiodate per mole of polysacchariderepeating unit. The serotype-specific charge of sodium metaperiodate wasselected to achieve a target level of Ps activation (moles aldehyde permole of Ps repeating unit).

For all serotypes, with the exception of serotypes 5 and 7F, theactivated product was diafiltered against 10 mM potassium phosphate, pH6.4, using a 10 kDa NMWCO tangential flow ultrafiltration membrane.Serotypes 5 and 7F were diafiltered against 10 mM sodium acetate, pH4-5. Ultrafiltration for all serotypes was conducted at 2-8° C.

Polysaccharide Conjugation to CRM₁₉₇

Oxidized polysaccharide solution was mixed with water and 1.5 Mpotassium phosphate, buffered at either pH 6.0 or pH 7.0, depending onthe serotype. The buffer pH was selected to optimize stability ofactivated Ps during the conjugation reaction. Purified CRM₁₉₇, obtainedthrough expression in Pseudomonas fluorescens as previously described(WO 2012/173876 A1), was 0.2-micron filtered and combined with thebuffered polysaccharide solution at a polysaccharide to CRM₁₉₇ massratio ranging from 0.4 to 1.0 w/w depending on the serotype. The massratio was selected to control the polysaccharide to CRM₁₉₇ ratio in theresulting conjugate. The polysaccharide and phosphate concentrationswere serotype-specific, ranging from 3.6 to 10.0 g/L and 100 to 150 mM,respectively, depending on the serotype. The serotype-specific Psconcentration was selected to control the size of the resultingconjugate. The solution was then 0.2-micron filtered. Nickel chloridewas added to approximately 2 mM using a 100 mM nickel chloride solution.Sodium cyanoborohydride (2 moles per mole of polysaccharide repeatingunit) was added. Conjugation proceeded for a serotype-specific duration(72 to 120 hours) in order to maximize consumption of Ps and protein.

Reduction with Sodium Borohydride

Following the conjugation reaction, the batch was diluted to a Psconcentration of approximately 3.5 g/L, cooled to 2-8° C., and1.2-micron filtered. All serotypes (except serotype 5) were diafilteredagainst 100 mM potassium phosphate, pH 7.0 at 2-8° C. using a 100 kDaNMWCO tangential flow ultrafiltration membrane. The batch, recovered inthe retentate, was then diluted to approximately 2.0 g Ps/L andpH-adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4.Sodium borohydride (1 mole per mole of polysaccharide repeating unit)was added. 1.5 M potassium phosphate, pH 6.0 was then added. Serotype 5was diafiltered against 300 mM sodium bicarbonate, pH 9, using a 100 kDaNMWCO tangential flow ultrafiltration membrane and then pH-neutralized.

Sterile Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDa NMWCOtangential flow ultrafiltration membrane. The retentate batch was0.2-micron filtered.

Serotype 19F was incubated for approximately 7 days at 22° C.,diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0 at4° C. using a 100 kDa NMWCO tangential flow ultrafiltration membrane,and 0.2-micron filtered.

The batch was adjusted to a Ps concentration of 1.0 g/L with additional10 mM histidine in 150 mM sodium chloride, pH 7.0. The batch wasdispensed into aliquots and frozen at ≤−60° C.

Example 3 Methods for the Conjugation of Serotypes 6A, 6B, 7F, 9V, 18C,19A, 19F, 22F, 23F and 33F to CRM₁₉₇ Using Reductive Amination inDimethylsulfoxide

The different serotype polysaccharides were individually conjugated tothe purified CRM₁₉₇ carrier protein using a common process flow.Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM₁₉₇ were individually lyophilized andredissolved in dimethylsulfoxide (DMSO). Redissolved polysaccharide andCRM₁₉₇ solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled toserotype-specific values in the sections below.

Polysaccharide Size Reduction and Activation

Purified pneumococcal capsular Ps powder was dissolved in water, and allserotypes, except serotype 19A, were 0.45-micron filtered. Allserotypes, except serotypes 18C and 19A, were homogenized to reduce themolecular mass of the Ps. Homogenization pressure and number of passesthrough the homogenizer were controlled to serotype-specific targets(150-1000 bar; 4-7 passes). Serotype 18C was size-reduced by acidhydrolysis at ≥90° C. Serotype 19A was not size-reduced.

Size-reduced polysaccharide was 0.2-micron filtered and thenconcentrated and diafiltered against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane wasused for serotype 18C.

The polysaccharide solution was then adjusted to a serotype-specifictemperature (4-22° C.) and pH (4-5) with a sodium acetate buffer.Polysaccharide activation was performed via periodate oxidation. Psactivation was initiated with the addition of a sodium metaperiodatesolution. The amount of sodium metaperiodate added wasserotype-specific, ranging from approximately 0.1 to 0.5 moles of sodiummetaperiodate per mole of polysaccharide repeating unit.

For all serotypes, the activated product was diafiltered against 10 mMpotassium phosphate, pH 6.4, and water using a 10 kDa NMWCO tangentialflow ultrafiltration membrane. A 5 kDa NMWCO membrane was used forserotype 18C. Ultrafiltration for all serotypes was conducted at 2-8° C.

Polysaccharide Conjugation to CRM₁₉₇

Purified CRM₁₉₇, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.0 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

The oxidized polysaccharide solution was formulated with water andsucrose in preparation for lyophilization. The protein solution wasformulated with water, phosphate buffer, and sucrose in preparation forlyophilization. Sucrose concentrations ranged from 1 to 5% to achieveoptimal redissolution in DMSO following lyophilization.

Formulated Ps and CRM₁₉₇ solutions were individually lyophilized.Lyophilized Ps and CRM₁₉₇ materials were redissolved in DMSO andcombined using a tee mixer. Sodium cyanoborohydride (1 moles per mole ofpolysaccharide repeating unit) was added, and conjugation proceeded fora serotype-specific duration (1 to 48 hours) to achieve a targetedconjugate size.

Reduction With Sodium Borohydride

Sodium borohydride (2 mole per mole of polysaccharide repeating unit)was added following the conjugation reaction. The batch was diluted into150 mM sodium chloride at approximately 4° C. Potassium phosphate bufferwas then added to neutralize the pH. The batch was concentrated anddiafiltered at approximately 4° C. against 150 mM sodium chloride usinga 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

Each batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDa NMWCOtangential flow ultrafiltration membrane. The retentate batch was0.2-micron filtered.

Serotype 19F was incubated for approximately 5 days at 22° C.,diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0 atapproximately 4° C. using a 300 kDa NMWCO tangential flowultrafiltration membrane, and 0.2-micron filtered.

The batch was diluted with additional 10 mM histidine in 150 mM sodiumchloride, pH 7.0 and dispensed into aliquots and frozen at ≤−60° C.

Example 4

In-Process Incubation of Serotype 19F-CRM₁₉₇ Conjugate for ImprovedConjugate Stability

During routine process development, drug product (DP) stability studiesat 4° C. using a serotype 19F-CRM₁₉₇ conjugate prepared as described inExample 3 without incubation (after the 300 kDa ultrafiltration, atfinal filtration and product storage) showed an initial rapid decline inserotype 19F potency with time (Table 1, conjugate lot A). The relativepotency decrease of approximately 20% from initial levels inapproximately 3 months revealed that the serotype 19F conjugate wasunstable. Note that no such trend was observed for other serotypes.Correspondingly, drug substance (DS) stability studies using the sameserotype 19F-CRM₁₉₇ conjugate showed an initial rapid increase in freepolysaccharide (Ps) content (FIGS. 1-2, conjugate lot A) and decrease inconjugate size (Tables 2-3, conjugate lot A), indicative of conjugatedPs loss. The initial rapid increase in free Ps was followed by aleveling off in free Ps content, suggests that there is a population oflabile sites within the serotype 19F conjugate. These labile sitesdegrade quickly under typical storage conditions liberating free Ps,reducing conjugate size, and resulting in potency loss. Eventually, whenlabile sites have degraded, the free Ps and potency changes with timeare significantly reduced.

TABLE 1 Relative potency, based on total polysaccharide ELISA results,for serotype 19F conjugate lots in DP formulations at 4° C. Serotype 19Fconjugate lot B was incubated at 22° C. for approximately 5 days in 10mM histidine, 150 mM sodium chloride, pH 7.0 prior to wide-poreultrafiltration purification and DP formulation. Serotype 19F conjugatelot A was not incubated during its production prior to DP formulation.Relative potency of serotype Relative potency of serotype Time at 19F inDP formulated using 19F in DP formulated using 2-8° C. non-incubatedserotype 19F incubated serotype 19F (months) conjugate lot A conjugatelot B 0 1.0 1.1 3 0.8 1.1

TABLE 2 Serotype 19F conjugate stability as measured by conjugate sizechange at 4° C. Conjugate size change for serotype 19F-CRM₁₉₇ conjugatelots A and B as a function of time at pH 7.0 and 4° C. Serotype 19Fconjugate lots A and B are described Table 1. Conjugate size (Mw) wasmeasured by high performance size exclusion chromatography with UVabsorbance, multi-angle light scattering, and refractive index detection(HPSEC UV-MALS-RI). Time at Conjugate size change, Conjugate sizechange, 2-8° C. non-incubated serotype 19F incubated serotype 19F(months) conjugate lot A conjugate lot B 6 −44% −2%

TABLE 3 Serotype 19F conjugate stability as measured by conjugate sizechange at 25° C. Conjugate size change for three serotype 19F- CRM₁₉₇conjugate lots as a function of time at pH 5.8 and 25° C. Serotype 19Fconjugate lots A and B are described Table 1; serotype 19F conjugate lotC is described in FIG. 2. Conjugate size (Mw) was measured by HPSECUV-MALS-RI. Conjugate size Conjugate size Conjugate size Time at change,non-incubated change, incubated change, incubated 25° C. serotype 19Fserotype 19F serotype 19F (weeks) conjugate lot A conjugate lot Bconjugate lot C 4 −29% +4% +2

Based on the results for serotype 19F lot A shown in FIGS. 1-2 andTables 1-2, an in-process incubation step was developed and incorporatedin the manufacturing DS process to improve the stability of serotype 19Fconjugate. Following the in-process incubation step, conjugates werepurified using a wide-pore ultrafiltration step to remove free Ps andlow molecular weight conjugates resulting from incubation. Thein-process incubation step was conducted after reduction with sodiumborohydride and allows liberation and removal of free Ps that wouldotherwise evolve over time in DS or DP. As described above, serotype 19Fconjugate lots B in and C in FIGS. 1-2 and Tables 1-2 had been incubatedduring their manufacture at pH 7.0 for approximately 5 days at 22° C. in150 mM sodium chloride with 10 mM histidine (lot B) or 25 mM potassiumphosphate (lot C). A comparison of the stability results for lots A tolots B or C shows that the in-process incubation significantly improvedthe stability of serotype 19F conjugate.

Actual free Ps profile during the in-process incubation of conjugatelots B and C is shown FIG. 3. Results show that free Ps contentimmediately started to increase during the incubation and laterplateaued at roughly 5 days.

In the absence of an incubation step, free Ps would be expected to begenerated at a similar rate in the drug substance and/or in the drugproduct leading to increased levels of free Ps in the vaccine.

A multivalent pneumococcal conjugate vaccine comprising serotype 19Fprepared according to the methods of the invention was found to beimmunogenic in mice, rabbits, non-human primates and in humans (data notshown).

1. A method for the production of a polysaccharide-protein conjugatecomprising Streptococcus pneumoniae serotype 19F capsular polysaccharidecovalently linked to a carrier protein from a mixture comprisingpolysaccharide-protein conjugate and free polysaccharide, the methodcomprising the steps of: a) incubating said mixture for a minimum of 6hours at a temperature ranging from 2-30° C. in a buffer having a pH inthe range of 5.0 to 9.0; and b) performing size separation underconditions that allow removal of free polysaccharide.
 2. The method ofclaim 1, wherein said size separation uses a nominal molecular weightcut off membrane of from 100 to 500 kDa whereby thepolysaccharide-protein conjugate is retained in the retentate.
 3. Themethod of claim 1, further comprising c) collecting thepolysaccharide-protein conjugate.
 4. The method of claim 1, wherein thecarrier protein is CRM₁₉₇.
 5. The method of claim 2, wherein theretained polysaccharide-protein conjugate has an average molecularweight of 600 kDa or more.
 6. The method of claim 5 wherein thepolysaccharide-protein conjugate has an average molecular weight of 1000kDa or more.
 7. The method of claim 1, wherein said buffer is selectedfrom a phosphate buffer, histidine, and TRIS.
 8. The method of claim 1,wherein said buffer has a pH in the range of pH 5.8 to 7.0.
 9. Themethod of claim 8, wherein said buffer has a pH of 7.0.
 10. The methodof claim 1, wherein the incubation temperature is in the range of 4-25°C.
 11. The method of claim 1, wherein said size separation is bysize-exclusion chromatography, bind/elute chromatography, or wide-poreultrafiltration.
 12. The method of claim 11, wherein said sizeseparation is by wide-pore ultrafiltration with a membrane having a MWCOof 100 kDa to 300 kDa.
 13. The method of claim 1, wherein the incubationis for at least 12 hours.
 14. The method of claim 13, wherein theincubation is for 108 to 132 hours.
 15. The method of claims 1, furthercomprising formulating the polysaccharide-protein conjugate with one ormore additional polysaccharide-protein conjugates from a differentserotype.
 16. The method of claim 15, further comprising formulatingwith an adjuvant.
 17. A serotype 19F polysaccharide protein conjugateproduced by the method of claims
 1. 18. A method for measuring doseand/or potency in a pneumococcal conjugate vaccine manufacturing lotcomprising performing an immunoassay for serotype 19F polysaccharideprotein conjugate in said manufacturing lot using the 19F conjugate ofclaim 17 as a reference standard.